You have all heard about the DNA double helix and genes. Many of you know that mutations occur randomly, that the DNA sequence is read by successive groups of three bases (the codons), that many genes encode enzymes, and that gene expression can be regulated.
These concepts were proposed on the basis of astute genetic experiments, as well as often on biochemical results. The original articles were these concepts appeared are however not frequently part of the normal curriculum of biologists, biochemists and medical students.
This course proposes to read study and discuss a small selection of these classical papers, and to put these landmarks in their historical context. Most of the authors displayed interesting personal histories and many of their contributions go beyond not only the papers we will read but probably all their scientific papers.
Our understanding of the scientific process, of the philosophy underlying the process of scientific discovery, and on the integration of new concepts is not only important for the history of science but also for the mental development of creative science.

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BS

Outstanding: the classic papers are always the best primary source of information!

SP

Apr 21, 2020

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Amazing course! Even more interested in molecular genetics. Best professor!

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Session 6

Benzer and Champe studied the properties of deletions that cover the boundary between rIIA and rIIB. As expected, most of them cannot provide either function during infection of a non-permissive strain. One deletion however was highly unusual and was still able to provide rIIB function even though it lacks 10% of the rIIB sites. This deletion was instrumental in confirming the general nature of the genetic code proposed by Crick et al. In the discussion, the authors evoke the notion of bi-functional enzymes such as tryptophane synthase. In bacteria, the two catalytic activities are performed by individual proteins encoded by adjacent enzymes. In eucaryotes, both reactions are performed by a single protein: the product of the first reaction does not diffuse out but is “funneled” into the second active site. This is just one exception to the one gene one enzyme model discussed in the first session.

Crick et al. start their paper by presenting the evidence that the code must be non-overlapping. One evidence, provided by Brenner, is the founding work of what will become bioinformatics. Starting with a single rIIB frameshift mutation, now known to involve the addition of a single base pair that displaces the reading frame of the mRNA, they isolate many intragenic suppressor mutations that restore rIIB function. The original mutation is given a + sign, and its suppressors a - sign. They then isolate suppressors of these suppressors that are themselves + mutants. All tested combinations of two + and two – mutations lack rIIB function. Most combinations of a + and a – mutations have rIIB function; the other combinations are proposed to generate a stop codon between the two frame shifts. Finally, a number of triple mutants were shown to have rIIB function. They use the unusual deletion that fuses the two rII genes to demonstrate that frameshift mutation located in the rIIA portion of the fused gene abolish its rIIB function. The simplest interpretation is that + mutants have one more (or one less) base pair and that – mutants have one less (or one more) base pair. Although the general nature of the code is 3n base pairs per amino acid, they belive that the code is a three rather than a six letters code.

Ministrado por

Dominique Belin

Transcrição

[MUSIC] These are the results of the complementation test. The complementation test is a test for function. The cripple and the blind. Which I used last week. So, what you see here, are a bunch of infections in a host that cannot allow an rII mutant to grow. As you see, all these what he called the self test, all of these are negative. There is no phage production. All of these are rII mutant. And the is making a lot of phage. And this is a typical complementation between an rIIA mutant and an rIIB mutant. The first genome on the top is providing the normal rIIB function. The bottom one is providing the normal rIIA function. So in the complementation test, Benzer and Champ used two rIIA deletions and these two show that 1589 is rIIA- as expected. But in the next one, all these five complementation tests give a positive result, that is, phage production. You may say that 220 is different from 260 or 300 in terms of or 360 or 280. No in terms of yield of particle per infected cell between 100 and 300 is a normally. In his case the yield were reasonably high, they were above 200. So you have a deletion that is destroying a part of the. A part of the rII locust, including a part of the B locust. And, that deletion is not affecting the rIIB function. Even those these beginning of the region, the beginning of the rIIB gene has sites for mutation. So then Benzer introduces the concept of sense and nonsense. Sense means a nucleotide sequence that can be translated into a protein, a regular codon. Nonsense means a sequence of bases that cannot be translated into an amino acid. That's nonsense and sense. So he realizes that. And he explains that in his mind, and we will see that next week, in his mind, this indicates that the genetic code is not completely degenerate. A degenerate code is a code where all the words have a meaning. Where all the codons, all the nucleotide sequences can be expressed into an amino acid into a protein. That's a fully degenerate code. At the end of this, Benzer realizes not only the implication for the code, but he also realizes the implication for evolution. This paper described the first protein chimera. Chimera protein. Now chimera proteins are very common. People use them all the time. That's the first chimera protein. A chimera between part of A and part of B. And so he thinks about protein structure in general and he says, well maybe if you can fuse two genes to make a single gene, maybe you can separate a gene to make two separate genes. And one of the examples discussed by Benzer is the case of the [INAUDIBLE]. The [INAUDIBLE] in e coli have a TrpDG followed by a TrpAG and so he proposes that maybe during evolution there was an original gene that was a complete BA, and that BA got split. And this is one of the way evolution could work. Well, it's nice proposal, but it turns out that the proposal is not entirely accurate because it was found later that in eukaryotes such as yeast or neurospora the order of the genes is reversed. And TrpA precedes TrpB. And the two Polypeptide chains, the two proteins are linked together by what is called a linker. This is a crystal structure of a eukaryotic tryptophan synthetase with the TrpA gene in yellow and the TrpB gene in blue. And this you can join. There's no way you can keep this structure and join B first, A next. Because the of Bs in this region, the end terminus of A is on the other side of the molecule. There's no way you could link them. I mean, you would require a huge linker to do that. Now when the sequence came out, it was realized that in fact the two genes are separated by two nucleotide. A stop codon, a nonsense codon, tga. And that this is one of the examples where one base here, the A serves two function. One nucleotide is used as part of the stop nonsense codon of TrpB and this same A residue is part of the start codon of TrpA. So the amount of information that can be stored in a single base is more than what was thought before. Now you may think, TrpA, TrpB, one enzyme, two enzymes, two enzymes with one gene? I mean, how is that compatible with the one gene, one enzyme model of melun taken that we've seen in the first session. That's the reaction carried out by TrpA and TrpB. You first remove a Glycerol 3-phosphate. You have an indoor residue and then you add this part, which makes it Trp with amino acid. Now this reaction is carried out by TrpA and this by TrpB. In a typical enzyme reaction, you would have this IGP entering the enzyme and indole coming out of the enzyme because it's a product. And indole going back into the active site of the second enzyme. Well in fact this is basically one enzyme. Because the indole is not never free, the indole is produced here and is pushed down this tunnel into the second active style. So it's basically one reaction. Although you can write it as two reactions, but it's still one enzyme.